Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

Abstract

The human keratinocyte cell line HaCaT expresses essentially all epidermal differentiation markers but exhibits deficiencies in tissue organization as surface transplants in nude mice and even more so in organotypic co-cultures with fibroblasts. Whereas tissue differentiation by normal keratinocytes(NEKs) is regulated by stromal interactions, this mechanism is impaired in HaCaT cells. This regulatory process is initiated by interleukin-1 (IL-1)release in keratinocytes, which induces expression of keratinocyte growth factor (KGF/FGF-7) and granulocyte macrophage-colony stimulating factor(GM-CSF) in fibroblasts. Production and release of IL-1 is very low and,consequently, expression of the fibroblast-derived growth factors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GMCSF, respectively, is inefficient to improve stratification and differentiation by HaCaT cells due to the low expression of their cognate receptors. More importantly, expression and release of the autocrine keratinocyte growth factor TGF-α is dramatically decreased in HaCaT cells. Addition of TGF- α or EGF stimulated HaCaT cell proliferation but, even more effectively, suppressed apoptosis, thus facilitating the formation of a regularly stratified epithelium. Furthermore, TGF-αenhanced the expression of the receptors for KGF and GM-CSF so that addition of these growth factors, or of their inducer IL-1, further improved epidermal tissue differentiation leading to in vitro skin equivalents comparable with cultures of NEKs. Thus, supplementing TGF-α normalized epidermal tissue regeneration by immortal HaCaT keratinocytes and their interaction with stromal cells so that regular skin equivalents are produced as standardized in vitro models.

A Stable Niche Supports Long-Term Maintenance of Human Epidermal Stem Cells in Organotypic Cultures

“…Since fibroblast-produced growth factors can regulate the growth and differentiation of the keratinocytes [28,29], we reasoned that the restricted maintenance of the epidermis in the collagen-OTCs could be due to the exhaustion of fibroblasts to provide the appropriate growth factors. To test this, we performed reverse transcription PCR for amphiregulin, fibroblast growth factor-2 (FGF-2), insulin-like growth factor 2, and transforming growth factor-␣.…”

Id proteins: Novel targets of activin action, which regulate epidermal homeostasis

“…Since HaCaT cells differentiate similarly to primary keratinocytes in threedimensional organotypic cultures (Schoop et al, 1999;Maas-Szabowski et al, 2003), we established such cultures using either vector-transfected or HaCaT-Id1-HA cells. Figure 6 shows staining of cultures from one representative HaCaT/neo and one HaCaT/Id1-HA clone.…”

“…The preparation of the dermal equivalent based on type I collagen hydrogels has been described previously (Smola et al, 1998;Stark et al, 1999;Maas-Szabowski et al, 2003). Briefly, 80% (vol.)…”

Self Organized Skin Equivalent by Epithelial and Fibroblast Cells on Polycaprolactone Electrospun Mat with Porous Fibers

“…21,22 It has also been shown that HaCaT cells can organize into intact epithelia in the presence of human or mouse fibroblasts. 23 The cells selected for developing organotypic skin equivalent using PPCL was human keratinocytes and rabbit hair follicle fibroblast cells. To the authors knowledge this is the first report on an organotypic system using HaCaT and rabbit hair follicle fibroblast cells.…”